Multiple drug resistance (MDR) has become an increasing problem in clinical therapy. Especially immuno-compromised persons suffer from severe secondary infections with multi resistant pathogens during hospital treatment. Prevalent are infections with the pathogenic yeasts of the genus Candida. In addition to secondary infections, MDR is a spreading problem in cancer chemotherapy, as cancerous cells are also developing MDR to chemotherapeutics. Both types of MDR are mainly caused by drug exporting transporters with a very broad substrate specificity, making the chemical treatment inefficient. The laborious development of new antibiotics and chemotherapeutics does not really solve the problem as the cells quickly gain resistance to the new drug. In the presence of drugs, the genes coding for the drug transporters are exceedingly expressed. This leads to a quick outward transport of the drugs that thus cannot reach their targets inside the cells anymore.
MDR is frequently due to the altered expression of an ATP-binding cassette (ABC) transporter (Wadkins, R. M. et al., Intl. Rev. Cytology 171:121-165 (1997)). Over one hundred ABC transporters have been identified in species ranging from Escherichia coli to humans (Higgins, C. F., Cell 82:693-696 (1995)). Prominent representatives of MDR pumps conferring multidrug resistance are in Saccharomyces cerevisiae Pdr5p, Snq2p, Yor1p (Balzi, E. et al., Journal of Biological Chemistry 269:2206-2214 (1994); Decottignies, A. et al., Journal of Biological Chemistry, 270:18150-18157 (1995); Katzmann, D. J. et al., Molecular and Cellular Biology, 15:6875-6883 (1995); Servos, J. et al., Molecular and General Genetics 236: 214-218 (1993)) in Candida albicans Cdr1p and Cdr2p (Prasad, R. et al., Current Genetics 4:320-329 (1995); Sanglard, D. et al., Microbiology 143:405-516 (1997)) and in Homo sapiens P-glycoprotein (Pgp) Mdr1 (Ueda, K. et al., Journal of Biological Chemistry 262:505-508 (1987)). Pdr5p is part of the well known multiple drug resistance (MDR) network of S. cerevisiae (also called pleiotropic drug resistance, PDR). Pgp provides one mechanism of possibly inhibiting resistance in tumor cells to chemotherapeutic agents (Senior, A. E. et al., FEBS Letters, 377:285-289 (1995); Abraham, E. H. et al., Proc. Natl. Acad. Sci. USA 90:312-316 (1993)).
To abolish this phenomenon and block these pumps, most recently inhibitors of these membrane proteins are provided in combination with antibiotics and chemotherapeutics. However, the inhibitors themselves become substrates of the pumps and thus, loose their inhibitory effect (Maki, N. et al., The Journal of Biological Chemistry 278:18132-18139 (2003); Smith, A. J. et al., Journal of the Natural Cancer Institute 90:1161-1166 (1998)).
An alternative approach, which underlies the present invention and which should solve these problems in therapy, is to inhibit the expression of the transporter genes. When the expression of the pump genes is suspended the therapeutics cannot be transported out of the cells. The inhibition of the expression can occur at different steps in the regulatory network, e.g. at the promoter level of the transporter genes, at the level of transcription factor(s) of the promoters or even at the promoter level of the transcription factor(s). In consequence, inhibition of any element of the MDR regulatory network would lead to (re)sensitised formerly resistant cells. Applying such an inhibitory substance in combination with not any more effective antibiotics or chemotherapeutics would restore their antibiotic or chemotherapeutic effect in currently resistant pathogens or tumour cells, respectively.
However, a reliable system to identify substances that specifically inhibit transcription factors and/or their promoters as well as the target promoters is not available. Especially, no technique is known which would enable to screen for such substances in a one-step-procedure.
Most of the known screening systems for regulatory effectors have been based on measuring the activity of the respective promoters by using appropriate reporter genes, e.g. coding for the green fluorescent protein (GFP) (Chalfie, M. et al., Science 263:802-805 (1994); Cormack, B. P. et al., Microbiology 143:303-311 (1997); Wiesner, C. et al., Nucleic Acids Research 80:e80 (2002); Barelle, C. J. et al., Yeast 21:333-340 (2004)), for the β-galactosidase (Leuker, C. E. et al., Molecular Genetics and Genomics 235:235-241 (1992)) or for the luciferase (Bronstein, I. et al., Analytical Biochemistry 219:169-181 (1994); Vieeites, J. M. et al., Yeast 10:1321-1327 (1994); Srikantha, T. et al., Journal of Bacteriology 178:121-129 (1996); Leskinen, P. et al., Yeast 20:1109-1113 (2003)). To measure the activity of promoters the micro-array technique has been prevailing (Wolfsberg, T. G. et al., Genome Research 8:775-792 (1999); Devaux, F. et al., FEBS Letters 498:140-144 (2001); Hikkel, I. et al., The Journal of Biological Chemistry 278:11427-11432 (2003); Li, T. et al., Circulation Research 93:1202-1209 (2003)). A common characteristic of all of these systems and methods is that the inhibition of the transcription results in a negative phenotype and that the quantification of the phenotype requires individual measurements. A negative phenotype does not necessarily result from inhibition of the expression of the reporter gene but can also result from an unspecific metabolic inhibition within the cell. The above mentioned screening systems can not distinguish between specific inhibition of the regulatory network or unspecific metabolic inhibition.
One screening system (Kozovská, Z. et al., Int. J. Antimicrobial Agents 22:284-290 (2003); Kozowska, Z. et al., Int. J. Antimicrob. Agents 24:386-392 (2004)) is based on a dominant lethal reporter gene that allows to distinguish between a specific and an unspecific inhibition. The reporter gene is expressed under the control of the promoter of a MDR conferring gene, which in turn is under control of a transcription factor, the expression of which is inducible. Only the inhibition of the promoter (or the gene product itself) allows for growth of the cell. In this respect, unspecific metabolic inhibition is not detected by the system as it would also not allow cell growth. Unfortunately, expression of the reporter gene is lethal for the cell. Thus, cloning of the test strain is very difficult. It is only possible if the reporter gene is not expressed and thus, the lethal gene product not present in the cell. This also explains the elaborate promoter construct. The choice of promoters to be screened is limited to those that have no (or very low) basal activity and are controlled by transcription factors. However, some of the genes conferring MDR possess very active promoters. For example, the CDR1 gene which is mainly responsible for C. albicans MDR, has a promoter which is quite active in C. albicans and S. cerevisiae, even without the presence of additional transcription factor(s).
This promoter could not be used in the screening system of Kozowska et al. Moreover, also promoters of the transcription factors cannot be involved into the screening system as transcription factors can be introduced into the system only under the control of an inducible promoter. Otherwise, growth of the test strain is not possible. However, the upstream elements of a regulatory network are especially interesting as target for inhibition since they control the expression of several different MDR-genes. Consequently, such a screening system is not suitable to search for prospective agents to combat multidrug resistance in pathogenic organisms or cancerous cells.
Thus, there is a need for a test system which allows the identification of inhibitors of transcription of transporters which confer MDR and which furthermore allows the study of any element of the regulatory network controlling said transcription in said test system.